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PAGE 22 DIAGENODE IDEAL CHIP-SEQ KIT FOR TRANSCRIPTION FACTORS USER MANUAL Antibody binding beads Protease inhibitors Negative ChIP Beads are in suspension. The provided beads are coated with protein A. Resuspend into a uniform suspension before each use. Bead storage. Store at 4°C. Do not freeze. Antibody binding capacity. ~ 10 μg / 30 μl Storage. Some inhibitors are unstable in solution. The provided P.I. mix should be kept frozen at -20°C, and thawed before use. Use non-immune IgG in the IP incubation mix. Use the non-immune IgG fraction from the same species the antibodies were produced in. Do not add antibody to the IP. Incubation with beads, which were not coated with antibodies antibodies could also be used as a negative ChIP control. Use a specifically blocked antibody in parallel. Use one antibody in ChIP and, the same antibody that is blocked with specific peptide. To specifically block an antibody: pre-incubate the antibody with saturating amounts of its epitope specific peptide for about 30 minutes at room temperature before use in the IP incubation mix. Use the blocked antibody as a negative control in parallel with the unblocked antibody. How many negative controls are necessary? If multiple antibodies - of the same species - are to be used with the same chromatin preparation then a single negative ChIP control is sufficient for all of the antibodies used. Why is my antibody not working in ChIP? Antibody-antigen recognition can be significantly affected by the cross-linking step resulting in loss of epitope accessibility and/or recognition. Which antibody should I use in ChIP? Use ChIP-grade antibodies. If not available, it is recommended to use several antibodies directed against different epitopes of the same protein. Verify that the antibodies can work directly in IP on fresh cell extracts. Also, when testing new antibodies, include known ChIP-grade antibodies as positive control for your ChIP assay. How do I choose an antibody for ChIP? Be aware of the possible cross-reactivity of antibodies. Verify by Western blot analysis the antibody specificity. Antigen affinity purification can be used to increase titer and specificity of polyclonal antibodies. control(s) There is a significant difference in affinity of different types of immunoglobulins to protein A or G. Thererfore, in function of the antibody used for your ChIP, it is recommended to choose either protein A or protein G coated beads. Species Immunglobulli Isotype Human Antibody in IP Protein A Protein G IgG1 +++ +++ IgG2 +++ +++ IgG3 - +++ IgG4 +++ +++ IgGM Are my antibodies going to bind the protein A or protein G? Mouse IgGF - + IgGA - + IgG1 IgG2a + +++ +++ +++ IgG2b ++ ++ IgG3 IgGM Rat IgG1 IgG2a IgG2b IgG2c Avoid multiple freeze/thawing. Innovating Epigenetic Solutions + + Use anti Mouse IgM + + +++ ++ ++ Chicken All Isotypes Cow All Isotypes ++ ++ +++ Goat All Isotypes Guinea Pig All Isotypes Hamster All Isotypes Horse All Isotypes Pig All Isotypes Rabbit All Isotypes +++ + ++ + +++ ++ ++ ++ +++ ++ ++ - ++ Sheep All Isotypes Freezing Use anti Human IgM Snap freeze and thaw on ice (e.g.: fixed cell pellets and sheared chromatin)
